The influence of mass spectrometry has emerged greatly due to its applications in genomics, proteomics and metabonomics. Matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) allows for the production of intact gas-phase ions of large non-volatile biomolecules. Several biological problems concerning the use of ESI-MS demand high-mass accuracy. These mass spectrometry techniques are disclosed generally in Mann et al., Analysis of Proteins and Proteomes By Mass Spectrometry, Annu. Rev. Biochem. 2001, 70:437-73, and Flora and Muddiman, High Mass Accuracy of Product Ions Produced by SORI-CID Using a Dual Electrospray Ionization Source Coupled with FTICR Mass Spectrometry, Analytical Chemistry, 2001, 73, 6, 1247-1251, both of which are incorporated herein by reference.
The measurement of a peptide's mass to within 1-2 ppm has been shown to uniquely identify the peptide and its source protein when the C-terminal amino acid is constrained to an arginine or lysine. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) has the ability to offer ≦1 ppm mass accuracy and has proven to be useful for protein identification in conjunction with protein databases. However, space-charge effects are known to profoundly influence the level of mass accuracy that can be achieved by FT-ICR-MS.
Accurate mass measurements using FT-ICR-MS depend on the ability to accurately measure an ion's cyclotron frequency while it is trapped in the homogeneous region of the magnetic field. Variations in magnetic field strength, trapping potentials, ion populations and excitation variables can produce changes in the cyclotron frequency that must be correctly compensated if accurate mass measurements are to be obtained. Efforts to account for these variables and increase the mass accuracy for FT-ICR-MS can essentially be divided into two general strategies: 1) external and 2) internal mass calibration.
External calibration, which relies on a calibration equation and a matching of total ion intensities for peaks of the analyte and the calibration spectra, has recently been shown to yield mass accuracies in the low ppm range. Another approach capitalized on the multiplicity of charge-states and minimized the mass error by systematically varying the frequency offset. Unfortunately, external calibration methods to account for total ion intensity can become tedious when a variety of ionic species results in a multiplicity of ion cloud distributions. Moreover, the use of a calibration equation based solely on the total ion intensity may be an over simplification. Regardless of these intricate arguments, it is generally well accepted that compensation for total ion intensity (i.e., variations in the electric field which perturb the frequency of the trapped ions in a linear fashion) is the dominant factor which must be taken into account to achieve high mass accuracy.
Internal calibration, also relying on a calibration equation, is based on measuring ion masses for the analyte and internal standard under identical conditions. Internal calibration is certainly a more straightforward approach because space charge effects, trapping, and detection factors are essentially identical for all species. The use of a dual electrospray ionization source to internally mass calibrate FT-ICR mass spectra of biological molecules, including calibrating tandem mass spectra, is disclosed generally in the Flora and Muddiman Analytical Chemistry article identified above and in Hannis and Muddiman, A Dual Electrospray Ionization Source Combined With Hexapole Accumulation to Achieve High Mass Accuracy of Biopolymers in Fourier Transform Ion Cyclotron Resonance Mass Spectroscopy, J. Am. Soc. Mass. Spectrom. 2000, 11 , 876-883, which is hereby incorporated by reference. This reported source was used for a wide variety of investigations. Separation of the internal calibrant from the analyte avoids preferential ionization and lends itself to coupling with on-line liquid separations. There are other reports, which utilized this general strategy with dual-ESI with FT-ICR and alternative mass analyzer technology to obtain high mass measurement accuracy each with its own advantages and disadvantages.
There remains, however, a continuing need for improved ESI sources. In particular, there is a need for ESI sources that are capable of accurately positioning the sample streams within time frames that are compatible with liquid separations. Any such source should also be capable of operating properly for extended periods of time.